Functional optical imaging encompasses the use of optical imaging techniques to study living biological systems in their native environments. Optical imaging techniques are well-suited for functional imaging because they are minimally-invasive, use non ionizing radiation, and derive contrast from a wide range of biological molecules. Modern transgenic labeling techniques, active and inactive exogenous agents, and intrinsic sources of contrast provide specific and dynamic markers of in-vivo processes at subcellular resolution. A central challenge in building functional optical imaging systems is to acquire data at high enough spatial and temporal resolutions to be able to resolve the in-vivo process(es) under study. This challenge is particularly highlighted within neuroscience where considerable effort in the field has focused on studying the structural and functional relationships within complete neurovascular units in the living brain. Many existing functional optical techniques are limited in meeting this challenge by their imaging geometries, light source(s), and/or hardware implementations. In this thesis we describe the design, construction, and application of novel 2D and 3D optical imaging systems to address this central challenge with a specific focus on functional neuroimaging applications. The 2D system is an ultra-fast, multispectral, wide-field imaging system capable of imaging 7.5 times faster than existing technologies. Its camera-first design allows for the fastest possible image acquisition rates because it is not limited by synchronization challenges that have hindered previous multispectral systems. We present the development of this system from a bench top instrument to a portable, low-cost, modular, open source, laptop based instrument. The constructed systems can acquire multispectral images at >75 frames per second with image resolutions up to 512 x 512 pixels. This increased speed means that spectral analysis more accurately reflects the instantaneous state of tissues and allows for significantly improved tracking of moving objects. We describe 3 quantitative applications of these systems to in-vivo research and clinical studies of cortical imaging and calcium signaling in stem cells. The design and source code of the portable system was released to the greater scientific community to help make high-speed, multispectral imaging more accessible to a larger number of dynamic imaging applications, and to foster further development of the software package. The second system we developed is an entirely new, high-speed, 3D fluorescence microscopy platform called Laser-Scanning Intersecting Plane Tomography (L-SIPT). L-SIPT uses a novel combination of light-sheet illumination and off-axis detection to provide en-face 3D imaging of samples. L-SIPT allows samples to move freely in their native environments, enabling a range of experiments not possible with previous 3D optical imaging techniques. The constructed system is capable of acquiring 3D images at rates >20 volumes per second (VPS) with volume resolutions of 1400 x 50 x 150 pixels, over a 200 fold increase over conventional laser scanning microscopes. Spatial resolution is set by choice of telescope design. We developed custom opto-mechanical components, computer raytracing models to guide system design and to characterize the technique's fundamental resolution limits, and phantoms and biological samples to refine the system's performance capabilities. We describe initial applications development of the system to image freely moving, transgenic Drosophila Melanogaster larvae, 3D calcium signaling and hemodynamics in transgenic and exogenously labeled rodent cortex in-vivo, and 3D calcium signaling in acute transgenic rodent cortical brain slices in-vitro.

The current work investigated the stimulation induced spatial response of the cerebral microvascular network by reconstruction of the 3D microvascular morphology from in vivo two photon fluorescence microscopy (2PFM) volumes using an automated, model based tracking algorithm. In vivo 2PFM imaging of the vasculature in the forelimb representation of the primary somatosensory cortex of alpha-chloralose anesthetized rats was achieved via implantation of a closed cranial window, and intravascular injection of fluorescent dextran. The dilatory and constrictory responses of the cerebral microvascular network to functional stimulation were heterogeneous and depended on resting vascular radius and response latency. Capillaries experienced large relative dilations and constrictions, but the larger vessel absolute volume changes dominated the overall network cerebral blood volume change.

The current work investigated the stimulation induced spatial response of the cerebral microvascular network by reconstruction of the 3D microvascular morphology from in vivo two photon fluorescence microscopy (2PFM) volumes using an automated, model based tracking algorithm. In vivo 2PFM imaging of the vasculature in the forelimb representation of the primary somatosensory cortex of alpha-chloralose anesthetized rats was achieved via implantation of a closed cranial window, and intravascular injection of fluorescent dextran. The dilatory and constrictory responses of the cerebral microvascular network to functional stimulation were heterogeneous and depended on resting vascular radius and response latency. Capillaries experienced large relative dilations and constrictions, but the larger vessel absolute volume changes dominated the overall network cerebral blood volume change.

A major contributor to relapse following smoking cessation is nicotine craving triggered by environmental cues, such as the sight of a lighted cigarette. Therefore, three integrated functional neuroimaging studies were conducted to examine the biological mechanisms underling cue-elicited craving for cigarettes. (1) First, I examined the effect of smoking-related pictorial cues on neural activation hi brain regions of interest (ROI) associated with reward signalling using functional magnetic resonance imaging (fMRI). Voxel-wise analysis demonstrated that smokers, but not nonsmokers, demonstrated significant activation associated with smoking-related pictorial cues in the anterior cingulate cortex, orbitofrontal cortex, and ventral striatum. Upon ROI analysis of the ventral striatum including the nucleus accumbens (VS/NAc), smokers exhibited significantly greater VS/NAc activation than non-smokers. (2) Next, I examined whether pre-specifled serotonergic polymorphisms would affect binding potential (BP) to a serotonin (5-HT) receptor implicated in the behavioural sensitisation process to nicotine (5-HTiA receptor). Healthy volunteers who had undergone positron emission tomography (PET) with a 5-HTiA-specific ligand [ U C]WAY-100635 were genotyped for the 5-HT₁ A -1018 G>C and 5-HT transporter (5-HTT) 5-HTT gene-linked polymorphic region (5-HTTLPR) polymorphisms. Participants carrying the 5-HTTLPR S allele (SS or SL genotypes) demonstrated significantly lower global presynaptic and postsynaptic BP compared to subjects with LL genotypes. (3) Finally, I triangulated the two initial studies to examine whether pre-specified trait (5- HTTLPR genotype) and/or state (smoking vs. abstinence) variables would influence cueelicited activation of the VS/NAc. There was greater activation to smoking-related cues in the VS/NAc of smokers during the smoking condition than the abstinent condition and a significant correlation between tobacco craving and VS/NAc activation in the smoking condition. The 5-HTTLPR polymorphism was not associated with VS/NAc activation. Power calculations are presented as the basis for future examination of genetic hypotheses. These data have implications for the ultimate goal of enhancing the efficacy of smoking cessation pharmacotherapy.

Cerebral small vessel disease (SVD) is associated with increased risks of stroke and dementia, however the mechanisms remain unclear. Low cerebral blood flow (CBF) has long been suggested and accepted, but clinical evidence is conflicting. On the other hand, growing evidence suggests that increased intracranial pulsatility due to vascular stiffening might be an alternative mechanism. Pulse-gated phase-contrast MRI is an imaging technique that allows measuring of CBF contemporaneously with pulsatility in multiple vessels and cerebrospinal fluid (CSF) spaces. The overall aim of this thesis was to provide an overview of existing clinical evidence on both hypotheses, to test the reproducibility of CBF and pulsatility measures in phase-contrast MRI, and to explore the relationship between CBF and intracranial pulsatility and SVD features in a group of patients with minor stroke and SVD changes on brain imaging. I first systematically reviewed and meta-analysed clinical studies that have assessed CBF or intracranial pulsatility in SVD patients. There were 38 studies (n=4006) on CBF and 27 (n=3356) on intracranial pulsatility. Most were cross-sectional, and longitudinal studies were scarce. There were large heterogeneities in patient characteristics and indices used particularly for measuring and calculating pulsatility. Methods to reduce bias such as blinding and the expertise of structural image readers were generally poorly reported, and many studies did not account for the impact of confounding factors (e.g. age, vascular risk factors and disease severity) on CBF or pulsatility. Evidence for falling CBF predating SVD was not supported by longitudinal studies; high pulsatility in one large artery such as internal carotid arteries (ICA) or middle cerebral arteries might be related to SVD, but studies that measured arteries, veins and CSF in the same patients were very limited and the reliability of some pulsatility measures, especially in CSF, needs to be tested. In order to test the reproducibility of the CBF and intracranial pulsatility measures, I repeated 2D phase-contrast MRI scans of vessels and CSF on healthy volunteers during two visits. I also compared the ICA pulsatility index derived from the MRI flow waveform to that from the Doppler ultrasound velocity waveform in patients with minor stroke and SVD features. In 10 heathy volunteers (age 35.2±9.78 years), the reproducibility of CBF and vascular pulsatility indices was good, with within-subject coefficients of variability (CV) less than 10%; whereas CSF flow and pulsatility measures were generally less reproducible (CV > 20%). In 56 patients (age 67.8±8.27 years), the ICA pulsatility indices in Doppler ultrasound and MRI were acceptably well-correlated (r=0.5, p < 0.001) considering the differences in the two techniques. We carried out a cross-sectional study aiming to recruit 60 patients with minor stroke and SVD features. We measured CBF and intracranial pulsatility using phase-contrast MRI, as well as aortic augmentation index (AIx) using a SphygmoCor device. I first investigated the relationship between intracranial measures, and systemic blood pressure or aortic AIx, and then focused on how the intracranial haemodynamic measures related to two main SVD features (white matter hyperintensities (WMH) and perivascular spaces (PVS)). We obtained usable data from 56/60 patients (age 67.8±8.27 years), reflecting a range of SVD burdens. After the adjustment for age, gender, and history of hypertension, higher pulsatility in the venous sinuses was associated with lower diastolic blood pressure and lower mean arterial pressure (e.g. diastolic blood pressure on straight sinus pulsatility index (PI): β=-0.005, P=0.029), but not with aortic AIx. Higher aortic AIx was associated with low ICA PI (β=-0.011, P=0.040). Increased pulsatility in the venous sinuses, not low CBF, was associated with greater WMH volume (e.g. superior sagittal sinus PI: β=1.29, P=0.005) and more basal ganglia PVS (e.g. odds ratio=1.379 per 0.1 increase in superior sagittal sinus PI) after the adjustment for age, gender and blood pressure. The thesis is the first to summarise the literature on CBF and intracranial pulsatility in SVD patients, addressed the major limitations of current clinical studies of SVD, and also assessed CBF and intracranial pulsatility contemporaneously in well-characterised patients with SVD features. The overall results of the thesis challenge the traditional hypothesis of the cause and effect between low CBF and SVD, and suggest that increased cerebrovascular pulsatility, which might be due to intrinsic cerebral small vessel pathologies rather than just aortic stiffness, is important for SVD. More importantly, this pilot study also provides a reliable methodology for measuring intracranial pulsatility using phase-contrast MRI for future longitudinal or larger multicentre studies, and shows that intracranial pulsatility could be used as a secondary outcome in clinical trials of SVD. However, future research is required to elucidate the implication of venous pulsatility and to fully explore the passage of pulse wave transmission in the brain. Overall, this thesis advances knowledge and suggest potential targets for future SVD studies in terms of mechanisms, prevention and treatment.

Electrical Impedance Tomography (EIT) is a technique for imaging the spatial distribution of conductivity inside a body using the boundary voltages, in response to applied current patterns, to reconstruct an image. Even though EIT has been proved useful in several medical applications such as mechanical respiration and ventilation monitoring of the lungs, its reported success in localising cerebral conductivity changes due to brain stimulation is very scant. In the case of the human head, the amplitude of the brain response to stimulation is usually very small and gets contaminated with physiological noise initiated from inside the cranium or the scalp. Three types of evoked responses were experimentally investigated: auditory startle response (ASR), CO2 reactivity response, and transient hyperaemic response (THR). ASR is expected to be a result of the brain’s functioning processes. However, the responses to CO2 and THR are expected to be due to cerebral blood volume or flow, due to physiological intervention in blood supply. According to the results, even when the amplitude of EIT measurements shows profound variation as in the case of CO2 reactivation, those could not be physiologically linked to the targeted responses and have been shown to be initiated from the scalp. The consistency of the measurements in the case of CO2 reactivation response was poor (37.50-50%). Meanwhile in the case of THR, although the magnitude of conductivity changes was overall 50% smaller than the previous cases, the subject movement was not necessary. This could be a reason that the consistency of THR case was very good (87%), and this can emphasize the necessity to maintain the changes in the scalp at minimum levels. In the case of ASR the response magnitude was very small (six times smaller than the CO2 reactivity case), and the evoked response can be detected with only 50% consistency. To measure very small EIT signals (such as those expected due to brain function) effectively, one must improve the sensitivity of the measurements to conductivity changes by increasing the excitation current. The functional EIT for Evoked Response (fEITER) system used in our investigations was modified from its initial configuration to increase its excitation current from 1 mApk-pk to 2 mApk-pk or 1 mArms. The bit-truncation in the process of Phase-Sensitive Detection (PSD) has also been improved, to modify the original 16-bit data readout to be 24-bit data readout. These improvements have doubled the instrument’s sensitivity, and have substantially reduced the truncation error to about 183 times. The quality of the physiological waveform was also significantly improved. Therefore, one could study more effectively very fast brain response using the modified system. For example, the latency of responses can be more precisely extracted, or the monitoring of the conductivity change in a period of only a few tens of milliseconds is then possible. The reconstruction of brain images corresponding to these physiologically evoked responses has been the ultimate goal of this thesis. To ensure obtaining the correct images, some crucial issues regarding EIT reconstruction were firstly investigated. One of these issues concerns the modelling error of the numerical head models. The reconstruction requires an accurate model capturing the geometry of the subject’s head with electrodes attached and accurate in-vivo tissue conductivities. However, since it is usually impractical to have a personalised model for each subject, many different head models (including a subject model) were constructed and investigated, to evaluate the possibility of using a generic model for all subjects. The electrode geometry was also carefully included into the models to minimise error. Another issue concerns the appropriate reconstruction algorithm. A novel nonlinear reconstruction method, based on the difference imaging approach and Generalized Minimal Residual method (GMRes) algorithm, with optimal parameters and prior information, was proposed to deal with significant modelling errors. With this algorithm, the experimental results showed that it is possible to use a generic model for reconstructing an impedance change, but the magnitude of the change should be rather small. The last issue tackled was regarding the a priori choice of model parameters, and in particular the tissue conductivities. The tissue conductivities of the scalp and the skull were also estimated by a proposed methodology based on the Gauss-Newton method. The estimation showed that, compared to previous reported values, the conductivity of the scalp was higher, at 0.58 S/m, and that of the skull lower, at 0.008 S/m. Eventually, by exploiting the hardware and firmware advances in the measuring instrument in conjunction with the proposed modelling and reconstruction algorithm, processing our experimental EIT data captured on human heads and a head-like tank confirm that the localisation and imaging of conductivity changes occurring within the head is indeed possible. From the low quality measurements in the case of the CO2 reactivity response, the reconstructed images of this response do not reflect the true conductivity change. The consistency of the images to localise the sources of the changes was very poor (0-50%), i.e. the conductivity changing locations in the images were likely to be random. Our analysis suggests that the changes inside the cranium are likely to be due to the large change in the scalp. In the case of THR, the reconstructed images were able to localise the response in a similar manner to what had been found on the measurements, and the consistency was quite high (76%). Meanwhile, in the case of ASR, surprisingly the consistency of the images was 82%, much higher than the consistency of the measurements, which was only 50%. This was because the changing amplitude of the measurements was too small to be noticed by visualisation, and it was practically cumbersome to investigate all measurements. This statistic confirms that image reconstruction can reveal information that is not directly apparent by observing the measurements. In summary, EIT can be used in brain (function) imaging applications to some extent. The targeted response, which typically originates from inside the cranium is always infused with neurophysiological noise or physical noise at the scalp, and the amplitude of noise determines the possibility to localise the changes. It is also necessary for the desired response to have sufficiently large amplitude. These results show that EIT has been successful in THR and ASR, but for CO2 reactivity response, EIT lacks the necessary sensitivity.

Testing facilitates memory retention more than studying. The current experiment aimed to investigate neural memory-related processes during repeated testing and studying, thereby contributing to more elaborate theories. 5 participants (aged 19-31) practiced word-pair associates during fMRI through repeated studying (40 pairs) and testing (40 pairs). One week later they returned for a test and the outcome was used to calculate subsequent memory differences during fMRI. Findings included higher subsequent memory difference in left parietal lobe, precuneus and superior frontal gyrus for the test condition, implying more elaborate semantic processing. Also, an interaction effect was found in anterior cingulate cortex, possibly indicating an early beneficial recruitment of memory enhancing functions in the test condition. / Testning förbättrar minnesinlagring mer än studium. Det aktuella experimentet syftade till att undersöka neurala minnesrelaterade processer under upprepad testning och studium, och därmed bidra till mer konkreta teorier. 5 deltagare (i åldern 19-31) tränade ordpar (Swahili-Svenska) i en fMRI-scanner, antingen genom upprepat studium (40 par) eller upprepad testning (40 par). En vecka senare återvände de för ett slutgiltigt test på alla ordpar. Resultatet användes för att beräkna skillnader mellan lyckade och ej lyckade omgångar i fMRI-scannern. Högre skillnader i vänstra inferiora parietalloben, precuneus och superiora frontalgyrus hittades för testning jämfört med studium, vilket kan spegla en djupare semantisk bearbetning. En interaktionseffekt hittades i anteriora cingulate cortex, vilket möjligen indikerar en tidig rekrytering av fördelaktiga minnesfunktioner i test-betingelsen.

Bipolar disorder (BD) and major depressive disorder (MDD) are characterised by a fundamental disturbance of mood, with strong support for overlapping causal pathways. Structural brain and neurocognitive abnormalities have been associated with mood disorders, but it is unknown whether these reflect early adverse effects predisposing to mood disorders or emerge as a consequence of illness onset. The Bipolar Family Study is well-suited to examine the origin of structural brain and neuropsychological abnormalities in mood disorders further. The volumes of subcortical brain regions, cortical thickness and surface area measures of frontal and temporal regions of interest and neuropsychological performance over a two-year time interval was compared at baseline and longitudinally between three groups: young individuals at high risk of mood disorders who subsequently developed MDD during the follow-up period (HR-MDD), individuals at high risk of mood disorders who remained well (HR-well), and healthy control subjects (HC). The longitudinal analysis of cortical thickness revealed significant group effects for the right parahippocampal and right fusiform gyrus. Cortical thickness in both of these brain regions across the two time points was reduced in both high-risk groups relative to controls, with the HR-MDD group displaying a thinner parahippocampus gyrus than the HR-well group. Moreover, a significant interaction effect was observed for the left inferior frontal and left precentral gyrus. The HR-well subjects had progressive thickness reductions in these brain regions relative to controls, while the HR-MDD group showed cortical thickening of these areas. Finally, longitudinal analyses of neuropsychological performance revealed a significant group effect for long delay verbal memory and extradimensional set-shifting performance. Reduced neurocognitive performance during both tasks across the two time points was found in the HR-well group relative to controls, with the HR-MDD group displaying decreased extradimensional set-shifting abilities as compared to the HC group only. These findings indicate, that reduced left parahippocampal and fusiform thickness constitute a familial trait marker for vulnerability to mood disorders and may thus form potential neuroanatomic endophenotypes. Particularly strong thickness reductions of the parahippocampal gyrus appear be linked to an onset of MDD. Moreover, progressive thickness reductions in the left inferior frontal and precentral gyrus in early adulthood form a familial trait marker for vulnerability to mood disorders, potentially reflecting early neurodegenerative processes. By contrast, an absence of cortical thinning of these brain regions in early adulthood appears to be linked to the onset of MDD, potentially reflecting a lack or delay of normal synaptic pruning processes. Reduced long delay verbal memory and extradimensional set-shifting performance across time constitute a familial trait marker for vulnerability to mood disorders, likely representing disturbances of normal brain development predisposing to illness. These findings advance our understanding of the origin of structural brain and neurocognitive abnormalities in mood disorders.

A central quest in biology is to understand the structure-function relationship of complex biological systems. The brain represents the ultimate complexity of a biological system: (1) the vertebrate brain contains 107-1011 neurons interconnected with glial cells; (2) over tens of diverse cell types are organized in a hierarchical way over an intricate landscape; (3) coordinated electrical and chemical activities of neuronal ensembles generate emergent properties and functions; and (4) each neuron can extend over large volumes with its spatial scales spanning 6 orders of magnitude. As a result, compared to other organ systems, our understanding of the brain remains primitive and obscure in terms of both its structures and its functions. Accordingly, many grand challenges endure in brain sciences, including comprehensively mapping neuronal wiring of the brain, an exhaustive taxonomy of cell types in the brain, and robust diagnostic and therapeutic strategies for brain diseases. These challenges are difficult to tackle with existing microscopy methods, because general trade-offs prevail between number of colors, imaging depth, spatial resolution, imaging throughput, sensitivity, and specificity. Therefore, the quest to understand the brain calls for advances and innovations on novel microscopy methods.The evolution of modern Raman microscopy is fundamentally driven by the development of novel spectroscopy methods. The advancement of molecular spectroscopy in turn pushes forward and benefits from, the progress in vibrational probes, labeling chemistry, and sample processing and transformation. In particular, stimulated Raman scattering (SRS) microscopy offers high sensitivity and fast acquisition for biomedical imaging, by harnessing accelerated vibrational transition from stimulated emission. Bio-orthogonal chemical imaging provides chemical specificity and minimal perturbation for visualizing metabolic dynamics of small molecules, by using tiny vibrational probes such as deuterium and alkyne. Electronic pre-resonance SRS (epr-SRS) microscopy further enhances the sensitivity to the nanomolar level for imaging specific proteins, by exploiting electronic pre-resonance of specially designed Raman dyes. Despite these notable innovations, the imaging depth of these Raman microscopy methods is limited to superficial layers of biological tissues (~100 μm) due to light scattering. This dissertation contributes to the development of advanced Raman microscopy methods for volumetric imaging with extended imaging depth in scattering tissues. For this purpose, we develop a set of tissue clearing strategies tailored to specific Raman imaging modalities. In addition, we develop image analysis methods to extract systems information from volumetric high-dimensional imaging datasets. Equipped with our volumetric imaging and analysis methods, we elucidate intricate structures and functions of the brain at both physiological and pathological conditions, providing implications for brain tumor metabolism and cerebellum development. Chapter 1 introduces an overview of Raman microscopy with particular emphasis on SRS and epr-SRS microscopies. Chapter 2 discusses the principles of tissue clearing with special focus on the basis of light scattering, the working mechanisms of different categories of tissue clearing methods, and the rationale underlying the development and evolution of these tissue clearing methods. Chapter 3 describes the development of volumetric chemical imaging, which brings label-free SRS microscopy, bio-orthogonal chemical imaging, and metabolic imaging to the realm of volumetric imaging with greater than 10-fold depth extension. Chapter 4 depicts the development of volumetric multiplex imaging, which generalizes epr-SRS microscopy to the territory of volumetric imaging. With this method we achieve one-shot imaging of more than 10 colors over millimeter-thick brain tissues, extending the imaging depth of multiplex protein imaging by 10~100 folds. Chapter 5 is a manuscript of an ongoing project on imaging nanocarriers for drug delivery across the blood-brain barrier (BBB). We develop a method of correlative multispectral SRS and fluorescence microscopy to image nanoparticles by SRS with multispectral information and particle counting capability and to image tissue context (especially cerebral vasculature) by fluorescence with high specificity. Using this method, we achieve direct imaging of nanocarriers that cross the BBB with definitive spectral evidence and single particle sensitivity. The preliminary results quantifying the proportion of nanoparticles that cross the BBB provide implications that challenge the current understanding of drug delivery to the brain.

The goal of this study is to determine the intentional movement direction based on the neural signals recorded from the motor cortex using optical brain imaging techniques. Towards this goal, we developed a cross-correlation analysis technique to determine the movement direction from the hemodynamic signals recorded from the motor cortex. Healthy human subjects were asked to perform a two-dimensional hand movement in two orthogonal directions while the hemodynamic signals were recorded from the motor cortex simultaneously with the movements. The movement directions were correlated with the hemodynamic signals to establish the cross-correlation patterns of firings among these neurons. Based on the specific cross-correlation patterns with respect to the different movement directions, we can distinguish the different intentional movement directions between front-back and right-left movements. This is based on the hypothesis that different movement directions can be determined by different cooperative firings among various groups of neurons. By identifying the different correlation patterns of brain activities with each group of neurons for each movement, we can decode the specific movement direction based on the hemodynamic signals. By developing such a computational method to decode movement direction, it can be used to control the direction of a wheelchair for paralyzed patients based on the changes in hemodynamic signals recorded using non-invasive optical imaging techniques.